1. Field of the Invention
The present application concerns molecular biology and more specifically the molecular components of the xe2x80x9cclockxe2x80x9d that times biological processes in green land plants.
2. Introduction and Related Art
Endogenous circadian rhythms exist in a wide variety of organisms both multicellular plants and animals as well as microorganisms. Circadian clocks regulating these rhythms consist of input pathways, a central oscillator and output pathways (14, 26, 48). Oscillators are thought to generate rhythms by a transcription-translation negative feedback loop (65, 16, 15, 64, 46). Studies in cyanobacteria, Neurospora, Drosophila and mouse have found that both positive and negative elements that activate and inhibit the transcription of clock genes are required to maintain the feedback loop (16, 15, 64, 46). In addition, postranscriptional and posttranslational regulation play an important role in circadian clocks in Drosophila and Neurospora (65, 51, 57). Input pathways from environmental cues such as light and temperature can entrain the oscillator, and it, in turn, regulates specific cellular events such as expression of clock-controlled genes (14, 26, 48). Until recently, little was known about circadian clocks in plants (33). In Arabidopsis thaliana, the toc1 mutant affects the period of many circadian rhythms (37, 52). Although the corresponding gene has not yet been cloned, it is thought that TOC1 encodes a component of the oscillator. The ELF3 gene has been proposed to act in the input pathway (23)
The phytochromes, a class of plant photoreceptors that has been extensively studied (44), regulate the expression of many genes, including the Lhcb genes which encode the chlorophyll a/b-proteins of photosystem II (59). A promoter region of the Lhcb1*3 gene of Arabidopsis thaliana that is essential for its regulation by phytochrome was identified (56, 27), and the CCA1 gene, whose product specifically interacts with this promoter region, was cloned (63). The CCA1 gene forms the subject of U.S. patent application Ser. No. 08/843572, filed on Apr. 18, 1997, which is incorporated herein by reference. The motif to which CCA1 binds is highly conserved in promoters of Lhcb genes from many species. Transgenic Arabidopsis plants expressing antisense CCA1 RNA showed reduced phytochrome induction of the endogenous Lhcb1*3 gene in etiolated seedlings. Furthermore, the increase in CCA1 mRNA in response to light preceded the increase in Lhcb1*3 mRNA (63). These data showed that CCA1 is a downstream component of the phytochrome signal transduction pathway leading to increased transcription of the Lhcb1*3 gene in Arabidopsis.
Expression of the Lhcb genes is also regulated by circadian rhythms (36). Characterization of CCA1 has shown that it is also involved in the circadian regulation of the Lhcb1*1 gene and in the control of other physiological rhythms, such as timing of flowering. CCA1 mRNA and protein levels themselves exhibit circadian oscillations, and overexpression of CCA1 repressed the expression of the endogenous CCA1 gene. Our earlier experimental results have demonstrated that the function of CCA1 is closely associated with the circadian oscillator itself (62). LHY, has also been identified as a potential clock genes (49). Constitutive expression of CCA1 was shown to abolish several distinct circadian rhythms and suppress its own expression as well as the rhythmic expression of LHY (61, 62). Lack of CCA1 in a T-DNA insertion mutant line shortened the periods of LHY and other clock-controlled genes (19). Overexpression of LHY also caused photoperiod insensitivity, arrhythmic expression of clock-controlled genes, and reduction of its own expression (49). These data suggest that both CCA1 and LHY may encode components of regulatory negative feedback loops closely associated with the central oscillator. The ESD4 (Early Short Days 4) gene of Arabidopsis is the subject of a patent publication (WO 98/56918) and has also been reported to alter responses to photoperiod.
To understand how CCA1 may function in the phytochrome signal transduction pathway and in the regulation of circadian rhythms, a yeast two-hybrid system was used to identify proteins that can interact with the CCA1 protein. A gene designated CKB3 whose product interacts specifically with CCA1 has been identified through use of the yeast two-hybrid system. CKB3 is a structural and functional homologue of the regulatory (xcex2) subunit of protein kinase CK2 in Arabidopsis. CK2 is a Ser/Thr kinase that is expressed ubiquitously and consists of two catalytic xcex1- and two regulatory xcex2-subunits. CKB3 and other xcex2-subunits of CK2 interact specifically with CCA1 both in the yeast two-hybrid system and in vitro. Recombinant CK2 can phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity, and that CK2 may play a role in the regulation of the circadian clock (55, 26, 48).
Recombinant plants that overexpress CKB3 were constructed. Overexpression of CKB3 resulted in increased CK2 activity and resulted in shorter periods of rhythmic expression of CCA1 and LHY, as well as of four other circadian clock-controlled genes. This resulted a significant shortening of time to flowering under short-day conditions. This change is flowering time was not accompanied by significant phenotypic changes in morphology. Alteration of CK2 activity, particularly through the overexpression of the CK xcex2-subunits represents a new and effective way of modulating flowering time in plants.